This invention relates to fluorescent compounds, their preparation and their use as labels in assay techniques.
Fluorescent compounds find wide application because of their ability to emit light upon excitation with energy within certain energy ranges. By virtue of this ability, fluorophores have found employment as labels in chemical or biological processes, e.g., assays. That is, various compounds can be conjugated to a fluorescent compound, the conjugate subjected to some type of partitioning, and the fate of the conjugate determined by irradiating the sample with light and detecting the zone in which the conjugate exists.
This technique can be employed, for example, in immunoassays involving specific binding pairs, such as DNA and like genomic material, ligands and receptors, e.g., antigens and antibodies. By conjugating a fluorophore to one of the members of the specific binding pair and employing various protocols, one can provide for partitioning of the fluorophore conjugate between a solid phase and a liquid phase in relation to the amount of antigen in an unknown sample. By measuring the fluorescence of either of the phases, one can then relate the level of fluorescence observed to a concentration of the antigen in the sample.
Alternatively, one can avoid partitioning of the fluorescent label by providing for a mechanism that varies the fluorescence of the label, depending upon the label environment in a liquid medium.
There is a particular interest in fluorescent compounds and assay techniques that permit multiple species to be labeled with different fluorophores. It is advantageous if the different fluorophores emit at distinguishably different wavelengths. It is also advantageous if the different fluorophores can be excited at a single wavelength. It is further desired to have fluorophores which are constructed to be chemically stable and to maintain their fluorescent conformation during use.
Photophysical characteristics of fluorophores play an important role in biological systems utilizing fluorescence detection techniques. The photographic industry has provided a number of fluorescent dyes with excellent photochemical properties in the form of high extinction coefficients, quantum yields and photostability. The cyanine, phthalocyanine, naphthalocyanine and squaraine classes of dyes have been used as sensitizers for photography and xerography in addition to being used as laser dyes, dyes for polymers, analytical indicators and fluorescent markers for biological macromolecules.
These commercially available dyes are typically hydrophobic and have to be dissolved in organic solvents prior to covalent attachment to proteins or nucleic acids which are usually in an aqueous media. This is a limitation to their use as fluorescent labeling reagents, as the requirement for an organic solvent or mixed solvent systems in which the biological macromolecules are only marginally soluble may result in poor labeling. Additionally, the organic solvents may be deleterious to the macromolecules. Post labeling, these hydrophobic dyes tend to xcfx80 stack in an aqueous environment which leads to fluorescence quenching. These hydrophobic dyes also bind nonspecifically to macromolecules by van der Waals and dipole-dipole type interactions. Such noncovalent attachment leads to instability and lower signal/noise ratio in assays employing these reagents.
The arylsulfonate cyanine dyes developed by Waggoner et al. (U.S. Pat. Nos. 5,569,766; 5,486,616; 5,268,486; 5,569,587 and 5,627,027) provide higher levels of solubility in aqueous media than similar hydrophobic materials. These dyes, with their sulfonate groups are also less prone to hydrophobic interactions such as stacking interactions when covalently bound to macromolecules. The latter effect leads to higher quantum yields in aqueous media and translates to a brighter labeling reagent, an important criteria for sensitive detection methods that use fluorescence. These arylsulfonate cyanine dyes also have functional groups appended to them for covalently coupling to macromolecules. Their absorption wavelength can be tailored to match existing instrumentation, which in turn increases their potential to be used as fluorophores for multiparameter analysis in cytometry and diagnostics.
Cyanine dyes have two heterocyclic moieties (typically two of the same heterocyclic moiety) covalently linked by a conjugated, unsaturated bridge. The extent of conjugation, the length of the linking chain and the nature of the heterocyclic groups determines the absorption wavelength.
The squaraine and the croconine dyes present a similar overall structure with the same types of heterocyclic groups linked through a conjugated, unsaturated chain. In the case of these materials, the linking chain includes an intervening cyclobuteneolate or cyclopenteneolate moiety.
The functional groups for linking these dyes to proteins are usually appended to the heterocyclic moiety by an appropriate linker. Two representative synthetic routes described in the art for forming these dyes and incorporating linking groups are provided in FIG. 1. Both routes go through a common intermediate, compound 3, which provides the heterocyclic moiety. In the case of the shorter route, 3 to 5 to 7, two molecules of 3 are linked to opposite ends of a precursor to the conjugated bridge. The presence of two heterocycles in each dye molecule prepared by this route therefore leads to a bisfunctional derivative such as the Cy7 bis-N-hydroxy succinimide ester 7, shown in FIG. 1. The bisfunctional derivatives provided by this synthetic route could cause the proteins to crosslink and in case of certain antibodies compromise their binding capacity. An alternative synthetic route, 3 to 4 to 6 to 8, is also shown in FIG. 1. This route employs a stepwise condensation of a functional-group-bearing heterocycle with an appropriate aldehyde. While it does yield the monofinctional derivative (as exemplified by the Cy5 mono N-hydroxy succinimide ester 6 synthesized in FIG. 1), the isolation and purification of intermediate 4, is problematic as some symmetrical (bisfunctional) dye is invariably produced. The length of the synthesis (five steps for monofunctional material versus three steps for bisfunctional), plus the tedious purification involved highlight the need for alternative methods for the synthesis of monofinctional cyanine and squaraine dyes that would be easier to carry out and would yield products that would be consequently easier to purify, as provided by the present invention.
The fluorescent dyes provided by the present invention include a second covalent link bridging the two heterocycles and restraining their motion relative to one another into conformations which favor fluorescent emission at wavelengths that are advantageous for use in assays. Other bridged cyanine dyes have been described in U.S. Pat. Nos. 5,571,388; 5,800,995; 4011,086; 3,904,637; 3,864,644; 3,821,233 and 4,490,463. Those in U.S. Pat. Nos. 5,571,388 and 5,800,995 have absorption and emission in the near to far infra red regions of the spectrum and lack functional groups necessary for water solubility. The materials shown in the earlier patents have rigidized monomethine or trimethine moieties linking the heterocycles. These materials absorb at lower wavelengths and also lack functional groups necessary for aqueous solubility, as well as, attachment to macromolecules.
A group of squaraine dyes have been described in U.S. Pat. No. 4,830,786 and subsequent divisional patents, U.S. Pat. Nos. 5,329,019; 5,416,214; 5,310,922 and 5,039,818. Assays for ligands and receptors employing conjugates of these dyes have been described in U.S. Pat. No. 4,806,488. Squaraine dyes with detergent-like properties have been used to stain cells in whole blood for the determination of blood group antigens. Squaraine dyes have also been used in fluorescent nucleic acid sequencing as described in WO 97/40104. Bridged squaraine dyes linked through nitrogens in the heterocyclic units are not known.
The present invention provides novel compounds in the squaraine and cyanine dye families. Squaraine and cyanine dyes share a common structure having two heterocyclic units conjugated to one another through an unsaturated linking chain. The compounds of the present invention are characterized by having a separate second linking chain joining the two heterocycles to restrain the compound into a desired conformation which absorbs electromagnetic energy at wavelengths greater than about 500 nm and especially greater than about 600 nm and emits fluorescence at wavelengths in the range of from about 640 nm to about 840 nm. This makes the compounds particularly suitable for excitation at such wavelengths such as the 633 nm wavelength of a helium/neon laser. A range of materials having a variety of fluorescence wavelengths are achieved.
In certain embodiments these compounds include one or more hydrophilic groups present to enhance water solubility. In other embodiments, they can be relatively oleophilic. Also, these compounds can include groups to facilitate their covalent attachment to proteins, nucleic acids and other biological and non-biological materials to make these materials fluoresce so that they can be detected in assays.
In another embodiment, this invention provides an improved method for preparing the bridged cyanine and squaraine dyes. In accord with this method, the two heterocycles present in both of these types of dyes are assembled prior to condensation with the conjugated unsaturated bridge by linking them together using an appropriate xe2x80x9csecondxe2x80x9d bridge which does not impart conjugation to the overall dye molecule and which may carry a functional group. This strategy yields monofunctional derivatives. Additionally, the facile intramolecular condensation involved in forming the dyes is an advantage over the less favorable intermolecular approach usually used.
In other embodiments, this invention provides the labeled materials which result when the dyes are attached to biological materials, methods of labeling and methods of analysis employing the labeled materials.
It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention as claimed.